1. Field of the Invention
The present invention is directed to DNA transformation constructs encoding mobile elements and their use for transforming eukaryotic cells. In particular transposons are used as a mechanism for inserting DNA sequences into the cell's genome after introduction of the transformation construct into the cell.
2. Description of the Related Art
Certain natural DNA sequences in eukaryotic and prokaryotic cells have the ability to move from one genomic locus to a second locus. These genetic elements are referred to generally as transposable elements or transposons. Advantageously, these transposable elements can be used as tools for genetically manipulating cells. In particular, transposable elements isolated from eukaryotes are anticipated as having the greatest potential for use in producing transgenic organisms.
Transposable elements can be divided into two classes. Class I are the retro-transposons that replicate through an RNA intermediate and utilize reverse transcriptase to produce a DNA molecule that is inserted into the host cell's genome. The Class II transposons include all other mobile elements and include P, hobo, mariner, Tcl, and Ac elements (Berg & Howe, Mobile DNA, American Society for Microbiology, Washington, D.C. 1989). Members of this transposon class have short inverted repeats at their termini and generate direct duplications of a host target sequence upon insertion. Many of these elements are currently being developed as general transformation vectors in insects and plants (Rubin & Spradling, Science, Volume 218, 348-353 1982; Lidholm, Lohe & Hartl, Genetics, Volume 134, 859-868 1993; O'Brochta & Handler, Prospects and possibilities for gene transfer techniques in insects, 451-488; in Molecular Approaches to Fundamental and Applied Entomology, ed. Oakeshott et al, Springer-Verlag, New York, 1993).
The P element has been used effectively for Drosophila transformation but has limited use as a general transformation vector because it is not active in species other than Drosophila (O'Brochta & Handler, 1993 supra; Rubin & Spradling, 1982 supra). The mariner element is phylogenetically dispersed (Robertson, H. Insect Physiol., Volume 41, 99-105, 1995), and therefore apparently has the capability of movement in a number of diverse species. In addition, the hobo element has demonstrated mobility in diverse genetic backgrounds and is a promising candidate for development as a genetic engineering tool (Atkinson, Warren & O'Brochta, PNAS USA, Volume 90, 9693-9697 1993; O'Brochta & Handler, 1993 supra; O'Brochta et al., Mol. Gen. Genet., Volume 244, 9-14, 1994).
PiggyBac (previously described as IFP2) and tagalong elements are unique Lepidopteran transposons structurally related to the Class II DNA transposable elements (Finnegan, Curr. Opin, Cell Bio., Volume 2, 471-477 1990). These transposons were isolated from the cabbage looper moth, Trichoplusia ni Hubner (Lepidoptera: Noctuidae). The piggyBac element was first identified as an insertion within Galleria mellonella or Autographa californica nuclear polyhedrosis virus genomes following passage of the viruses in the Trichoplusia ni insect cell line, TN-368 (Fraser et al., Virology, Volume 145, 356-361, 1985; Fraser et al., J. Virology, Volume 47, 287-300, 1983).
The piggyBac and tagalong elements are unusual among Class II transposons in that those elements always target and duplicate the tetranucleotide, TTAA, upon insertion in Baculovirus-infected cells (Cary et al., Virology, Volume 172, 156-169, 1989). The specificity for TTAA target sites is exhibited by other Lepidopteran transposon-like insertions as well (Beames & Summers, Virology, Volume 162, 206-220 1988; Beames & Summers, Virology, Volume 174, 354-363 1990; Carstens, Virology, Volume 161, 8-17, 1987; Oellig et al., J. Virology, Volume 61, 3048-3057, 1987; Schetter, Oellig & Doerfler, J. Virology, Volume 64, 1844-1850, 1990). Thus the piggyBac and tagalong elements are part of a subclass of the Class II transposons.
In addition to TTAA target specificity, all Lepidopteran transposons having the TTAA target specificity terminate in at least two C residues at the 5' ends of their inverted repeats. Given their similarity in insertion site selection and duplication, all of these TTAA specific elements are likely to excise in a similar manner.
Furthermore piggyBac and tagalong elements excise precisely upon transposition in vivo, leaving behind the single TTAA target sequence upon excision. The excision events of piggyBac and tagalong are dissimilar to the transposase-associated excision events of the hAT family of transposons. This family includes hobo, hermes, Ac and Tam3 (Calvi et al., Cell, Volume 66, 465-471, 1991). Elements in the hAT family vary in the length and nucleotide sequence of their inverted terminal repeats (Calvi et al., 1991; supra), but have a conserved A.sub.2 G.sub.5 motif within these repeats, and generate 8 bp target site duplications (Warren et al., Genet. Research, Volume 64, 87-97, 1994). These elements excise imprecisely in the presence of an element-encoded transposase and leave behind characteristic footprints that have proven useful in distinguishing transposase-associated excision events (Atkinson et al., 1993 supra; Warren et al., 1994 supra).
Most of the transposase-associated excisions of P-elements are imprecise events, leaving behind part or all of the 31 bp terminal inverted repeat and adding `filler` sequences at the excision breakpoints (O'Brochta et al, Mol. Gen. Genet., Volume 225, 387-394, 1991: Takasu-Ishikawa et al., Mol. Gen. Genet., Volume 232, 17-23, 1992). In the case of the hobo element of Drosophila melanogaster, excision from plasmids in microinjected fertile eggs most often involves the complete removal of hobo and some flanking nucleotides with the addition of filler sequences related to flanking host DNA at the excision breakpoints (Atkinson, Warren & O'Brochta, 1993 supra; Handler & Gomez, Mol. Gen. Genet., Volume 247, 399-408 1995; O'Brochta & Handler, 1993 supra).
In contrast with these other Class II elements, precise excision of piggyBac and tagalong is the rule rather than the exception. Precise excision of genetically tagged piggyBac elements was first demonstrated in Baculovirus genomes of infected cells (Fraser et al, Virology 211, 397-407 1995). However, the precise excision of the piggyBac element has also been demonstrated in non-virus infected cells indicating the excision of piggyBac is not dependent on Baculovirus protein products. The frequency of precise excision events in transiently transfected IPLB-SF21AE cells is greatly enhanced by the presence of a helper element encoding a full-length transposase. The excision event is believed to be a non-conservative event involving double-strand breaks at or near the transposon termini.
The present invention, discussed below, provides recombinant DNA vectors derived from the piggyBac and tagalong transposons which are different from related art vectors. Furthermore, the present invention provides a method to produce transgenic organisms using the recombinant DNA vectors. The transposon genetic transformation system of the present invention provides vectors and broad spectrum methods for the introduction of foreign genes that do not currently exist.